Hexokinase isoforms I and II bind to mitochondrial outer membranes in large part by interacting with the outer membrane voltage-dependent anion channel (VDAC). This interaction results in a shift in the susceptibility of mitochondria to pro-apoptotic signals that are mediated through Bcl2-family proteins. The upregulation of hexokinase II expression in tumor cells is thought to provide both a metabolic benefit and an apoptosis suppressive capacity that gives the cell a growth advantage and increases its resistance to chemotherapy. However, the mechanisms responsible for the anti-apoptotic effect of hexokinase binding and its regulation remain poorly understood. We hypothesize that hexokinase competes with Bcl2 family proteins for binding to VDAC to influence the balance of pro-and anti-apoptotic proteins that control outer membrane permeabilization. Hexokinase binding to VDAC is regulated by protein kinases, notably glycogen synthase kinase (GSK)-3beta and protein kinase C (PKC)-epsilon. In addition, there is evidence that the cholesterol content of the mitochondrial membranes may contribute to the regulation of hexokinase binding. At the same time, VDAC associated proteins are critically involved in the regulation of cholesterol uptake. A better characterization of these regulatory processes is required to elucidate the role of hexokinases in normal tissue function and to apply these insights for optimizing cancer treatment.